Lupus nephritis (LN) affects half of all systemic lupus erythematosus (SLE) patients. Among African-American women, a growing demographic in the Veteran population, this disease leads to 50% renal failure rate by five years. Effective LN drug development is hindered by the heterogeneity mechanisms leading to LN. Endothelial cells act as a final common dampener on inflammatory responses of many types through the action of endothelial nitric oxide synthase (eNOS) nitric oxide (NO). Lupus is characterized by dysfunction of these cells (endothelial cell dysfunction or ECD). This dysfunction leads to tissue inflammation such as proliferative lupus nephritis. Knowledge of mechanisms leading to ECD is essential to progress in developing novel and more broadly effective therapies for LN. Preliminary studies suggest that abnormal phosphorylation of eNOS at threonine 495 (Thr-495) and uncoupling of eNOS homodimers leads to ECD in SLE. The goal of this proposal is to define and target the common intracellular mechanisms leading to functional ECD and determine, in a discovery analysis, serum factors that signal for ECD in LN. To achieve this goal, the following aims are proposed: 1) Characterize intracellular pathways of eNOS dysfunction induced by SLE and LN ECD serum. We hypothesize that SLE serum induces ECD through the common final intracellular mechanisms of increased Thr-495 phosphorylation and eNOS dimer uncoupling. To address this hypothesis, serum samples from SLE, LN, and healthy control patients will be phenotyped for their ability to induce neutrophil migration and adhesion to cultured EC (in vitro ECD). eNOS and phospho eNOS protein expression and dimer coupling will be determined after culture with serum from these groups. 2) Assess the effect of agents that target pathways of eNOS dysfunction on SLE serum-induced in vitro ECD. We hypothesize that ECD induced by SLE serum can be reversed with agents designed to restore eNOS coupling, inhibit PKC-mediated Thr-495 phospho-eNOS or mimic eNOS NO. This aim will determine the ability of each of these approaches to restore endothelial function in cells cultured with ECD-inducing serum. Agents that target mechanism-based ECD will be added to culture with ECD-inducing SLE serum to test the effect on ECD. Pharmacodynamic markers will be used to determine optimal concentrations and mechanism-based action of each agent in association with the desired effect on EC function. 3) Discovery analysis of serum proteins and exosome lipids with differential expression in ECD serum. Preliminary studies suggest that lupus immunoglobulin fractions and exosomes induce ECD. Preliminary discovery analysis demonstrates that glycosphingolipids in exosomes associate with poor response to therapy. ECD and non-ECD LN, SLE, and control serum will be fractionated into exosome, immunoglobulin, and non-immunoglobulin, non-exosome fractions and tested for in vitro ECD- induction. Proteins unique to the above fractions that induce ECD will be determined by liquid chromatography and time-of-flight/time-of-flight mass spectrometry. Exosome lipids unique to ECD serum will be assayed by liquid chromatography and mass spectrometry. Pathway analysis will suggest pathways of ECD among molecules identified from ECD fractions. This project will fill gaps in our knowledge of how ECD induces pathologic inflammatory infiltrates in SLE and accelerate targeted therapies for Veterans with SLE. This study could accelerate development of therapies for diseases with ECD such as hypertension, atherosclerosis, and diabetic renal disease in Veterans.